Ciliated muconodular papillary tumor (CMPT) is a rare pulmonary tumor originating from the peripheral bronchial epithelium, characterized by papillary structures and extracellular mucin. It consists of a dual-layered epithelial structure composed of ciliated cells, mucous cells, and continuous basal cells. Therefore, this non-dysplastic and non-invasive nodular growth tumor is named CMPT (1). However, in 2018, Chang et al. (2) reported cases lacking papillary structures, cilia, and mucous cells. Nevertheless, these lesions had bilayered cell structures containing continuous basal cell layers; as such, they used bronchiolar adenoma (BA) as a broader term for their lesions. Based on histomorphological and immunohistochemical findings, two types of BA have been identified: proximal-type, containing ciliated and mucous cells with papillary to flat architectural patterns, and distal-type, containing clara cells and type II pneumocytes, with a flat pattern (2–4). Complete diagnosis depends on differential diagnosis from mucinous adenocarcinoma (5, 6), particularly in frozen section diagnosis during surgery. Recently, there has been an extensive discussion on whether BA has malignant potential, and it has been concluded that the risk of malignant transformation is minimal. The Fifth Edition WHO for thoracic tumors classified it as a benign tumor, with an ICD-O code of 8140/0. In this paper, we present a rare case of malignant transformation of BA to invasive mucinous adenocarcinoma (IMA). Genetic analysis of the KRAS and CDK6 driver genes revealed the same mutations in the BA region and IMA region of this lesion. The patient was followed-up for six months without any recurrence.

A 59-year-old man underwent thoracoscopic wedge resection of the right lower lobe on September 14, 2021. This procedure was conducted due to the presence of nodular hyperdense lesions in the subpleural region of the outer basal segment of the right lower lobe. The patient had undergone several chest CT scans over a period of four years from 2017, during which the nodule increased from 0.53 cm to 1.1 cm. The CT scans showed the gradual appearance of a marginal burr sign and central solid component over time ( Figures 1A–D , red arrow). The patient’s serum tumor markers, including carcinoembryonic antigen (CEA), cancer antigens 125 (CA125), cytokeratin 19-fragments (CYFRA21-1), and squamous cell carcinoma antigen (SCC), were all within normal limits except for neuron specific enolase (NSE), which was observed to be elevated to a level of 30ng/ml. During intraoperative gross examination, a gray-white nodule, with a maximum cross-sectional area of 0.8 x 0.5 cm, was observed immediately adjacent to the pleura. Preliminary diagnosis based on frozen sections was bronchioloalveolar carcinoma (BA). However, microscopic observation revealed the presence of a bilayered cell structure transitioning into tall columnar mucinous epithelium ( Figures 2A, B ), indicating the possibility of well-differentiated mucinous adenocarcinoma. A paraffin section of postoperative hematoxylin-eosin staining showed an unclear tumor boundary and a papillary structure with a skip growth pattern ( Figure 2C ). Within this nodule, extracellular mucus with a continuous basal cell layer, as well as a bilayered structure composed of ciliated cells or mucous cells growing along the alveolar wall, were observed ( Figure 2D ). However, some glands were lined with proliferative mucinous cells, with an area of 0.4 x 0.3cm2. Tall columnar mucous cells lacked cilia structures and were slightly atypical, with enlarged nuclei, an increased ratio of nucleus to cytoplasm, not easily visible mitotic figures and loss of basal cell layer ( Figures 2E, F ). Hence, immunohistochemistry was performed with a panel including CK7, TTF-1, Napsin A, CK5/6, p63, p40, CEA and Ki-67. Immunohistochemical expression showed that P40 and P63 markers were continuously expressed in the part of BA, whereas the positive expression of P40 and P63 was lost in the mucoid glandular structure ( Figures 3A–D ). The expression pattern of CEA was similar to that of P40 and P63 ( Figures 3E, F ). TTF-1 was positively expressed in type II alveolar epithelium and clara cells. In this case, TTF1 was expressed punctiformly in the glands of BA, but continuously and strongly positively in glands formed by highly columnar mucous cells ( Figures 3G, H ). As a result of these findings, the final diagnosis was determined to be BA, with malignant transformation into well-differentiated mucinous adenocarcinoma considered in some areas.

To further investigate the molecular mechanisms present in BA and IMA lesions, as well as the potential correlation between them, NGS sequencing was utilized. Specifically, a panel covering 116 genes was used for analysis of the regions of BA and IMA.

All tissue collections and experiments were reviewed and approved by the Biomedical Research Ethic Committee of Shandong Provincial Hospital (NO. SWYX2024-542). The study was conducted in accordance with recognized ethical guidelines (Declaration of Helsinki). Informed consent was obtained from all of the participants.

The sample type tested in our experiment was the FFPE sample. Before performing NGS, the paraffin section of postoperative hematoxylin-eosin staining was used for annotation of the IMA region and BA region. The tumor cell content in each area both reached 70%. Corresponding areas in 10 slides of 5 μm thick paraffin sections were scraped with a knife to obtain IMA and BA tissues, respectively. DNA and RNA were extracted from IMA and BA tissues using the FFPE DNA/RNA kit (Amoy Diagnostics Co., Ltd. Xiamen). The library construction was conducted with the Human Cancer Multigene Mutation Detection kit (8.06.0056, Amoy Diagnostics Co., Ltd. Xiamen). This kit contains RNA gene fusion and DNA gene mutation detection systems. A total of 50ng RNA were used for reverse transcription with MiniAmpTM Thermal Cycler (Thermo Fisher Scientific Inc). A total of 100ng DNA/cDNA were used for hybridization with probes to specifically capture the target region. The captured fragments were enriched by extension, ligation, enzyme digestion, amplification and magnetic bead purification by Agencourt AMPure XP Kit (Beckman Coulter) to obtain the sequenced library. Quality control of sequencing libraries was performed with the 2100 Bioanalyzer system with matching reagents (Agilent) and the QuantusTM Fluorometer series with matching reagents (Promega). The library was sequenced using ADx-SEQ200 Plus (Amoy Diagnostics Co., Ltd. Xiamen). After Sequencing was completed, data were analyzed with the Illumina Sequencing Analysis Viewer, which yielded a Q30 base ratio of 86%. The average effective sequencing depth of library DNA was 1616×, and the RNA-Control was 7148×. The threshold for hot spot mutations was set at ≥0.5% with an alteration depth of ≥4, while for non-hot spot mutations, the threshold was ≥3% with the same alteration depth requirement. The copy number alteration (CNA) threshold for DNA was set at ≥3.5 copies, and the microsatellite instability (MSI) ratio threshold was set at ≥15%. For RNA fusions, the threshold was ≥10 copies, and for MET exon 14 skipping, it was ≥40 copies.

Of significant note, KRAS mutations (p.G12V, c.35G > T, Exon2) were found in both portions, although the abundance of these mutations differed. These results suggest that exists a close association between driver gene mutations and the process of malignant transformation of BA into IMA. Additionally, a mutation in the cyclin-dependent kinases (CDK) 6 gene was identified in the lesions, a cell cycle gene with a mutation abundance of 51.45% in IMA and 49.01% in BA (refer to Table 1 ). Recognition of the role played by CDK6 as a cyclin D binding partner that controls the G1 phase and drives the cell cycle into the S phase is essential. Abemaciclib and Palbociclib can be used as the first selective inhibitor of CDK6 (7, 8).

Ciliated muconodular papillary tumor (CMPT) is a rare pulmonary tumor originating from the peripheral bronchial epithelium, characterized by papillary structures and extracellular mucin. It consists of a dual-layered epithelial structure composed of ciliated cells, mucous cells, and continuous basal cells. Therefore, this non-dysplastic and non-invasive nodular growth tumor is named CMPT (1). In 2018, Chang et al. (2) analyzed 25 cases of proliferative nodules that arise from bronchiolar epithelium, with only four cases meeting the CMPT description. They discovered 17 cases of morphologically identical distal respiratory bronchiolar epithelium comprising flat structures with loss of ciliated and mucinous cells, replaced by apical cuboidal cells and clara cells with cytoplasmic snouts structures. Despite this, double epithelial structures containing continuous basal cell layers formed both types of nodules, with micropapillary tufts generated by ciliated cells present in the alveolar space. As a result, they summarized two types of BA: proximal-type and distal-type using the BA concept. The primary objective of considering this classification was to aid in diagnosing rather than categorizing each tumor into a specific proximal or distal pattern. BA malignant transformation is rare, with only three reported cases ( Table 2 ). This case report describes a 59-year-old patient whose BA transformed malignantly into IMA.

BA is characterized by pure ground-glass and subsolid lesions in CT scans, with ill-defined peripheral opacity attributed to inflammatory infiltrates; lobular/spiculated margins indicate the probability of malignant invasion (9).

Distinguishing BA/CMPT from IMA in the frozen section is challenging. Although the frozen section reveals an area with glandular structures, consisting of a single layer of tall columnar mucous cells that lack obvious cellular atypia, the limitations of the frozen section make it difficult to differentiate between BA and IMA. Microscopic histomorphological features and the presence of basal cells are the only way to confirm the diagnosis. Diagnosis for cases with partial malignant transformation of BA is even more challenging. However, Intraoperative direct immunohistochemistry using basal cell markers such as P40, P63, or CK5/6 (10) can aid in diagnosing BA with malignant transformation to IMA during the frozen section. Moreover, postoperative paraffin sections revealed that basal cell loss in IMA is an important differential factor between BA and IMA that can be identified using immunohistochemical methods to label basal cells, such as P40, P63, or CK5/6, or TTF-1 for cuboidal cells not expressed in the mucous tall columnar cells of IMA. Liu et al. (11) suggests that BA may have carcinogenic potential, based on their use of a histological and immunohistochemical combination to diagnose a patient with BA accompanied by IMA.

Shao J (12) conducted a study where they sequenced 422 genes in 25 different samples. The study identified commonly mutated driver genes for BA, which include EGFR (52%), KRAS (16%), ERBB2 (8%), BRAF (12%), and RET fusion (4%), which were consistent with the BA driver gene mutations reported by Chang (2) and other studies. These mutations are found in high frequency in BA driver genes, and combined with a skip growth pattern and characteristic micropapillary structure, there has been recent debate about BA’s potential as a precursor lesion (5, 6, 13, 14). In this particular case, we report the presence of KRAS mutations (G12V) in both the BA and IMA regions. In Chang’s study (15), it was observed that KRAS mutations (76%) and specific gene fusions frequently characterized the IMA genome, with the G12V mutation being the second most common mutation (32%) after G12D (36%). Moreover, KRAS mutations have been reported in 24% of BA (6). As such, we speculate that in this case, certain parts of the lesion converted gradually from a BA to an IMA due to KRAS mutations, which was also observed on the imaging as an increasing density of nodule. Supporting the argument of BA as a precursor lesion, Han (6) found that sequencing analysis of the BA and IMA regions showed that the occurrence of KRAS mutation (G12V) in both regions infers that the IMA originates from BA, malignant transformation of BA to IMA. Li (14) reported a case of CCNE1 (Exon7, c.476A > G) mutation in mixed GGN and pure GGN, with mutation abundances of 48.83% and 45.41%, respectively. Overexpression of CCNE1 has been shown to be a poor prognostic indicator in lung cancer and to contribute to the growth and metastasis of the disease (16). In this study, the discovery of mutations in cell cycle genes CDK6 and its homolog CDK4 in both the BA and IMA regions was particularly noteworthy, with mutation abundance being higher than that of KRAS, reaching up to 49.01% and 51.45%. CDK6 specifically regulates the transcription of tumor-related genes such as vascular endothelial growth factor-A (VEGF-A) and early growth response gene-1 (EGR1) (7, 8), thereby promoting the transcription of genes from G1 to S phase. Yu and colleagues (17) identified CDK6 as one of the mRNAs differentially expressed in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) through mRNA and circRNA expression patterns comparison. Their analysis of expression profile data and clinical information of the two subtypes from TCGA concluded that overexpression of CDK6 predicted a poor prognosis in LUAD. Induction of CDK6 by tripartite motif-containing 59 (TRIM59) contributes to the epithelial-to-mesenchymal transition (EMT) process, while CDK4/6 mutations are associated with a poor prognosis in KRAS-mutant non-small-cell lung carcinoma (NSCLC), highlighting the role of CDK4/6 mutations as oncogenic factors that promote tumor growth and metastasis (17, 18). Importantly, in this case, the variant allele frequency (VAF) of CDK6 was higher in the IMA region than in the BA, further supporting the evidence of malignant transformation of the BA into IMA.

In summary, we report a rare case of malignant transformation of BA into IMA with identified mutations in KRAS and CDK6. This case provides histological and genetic evidence supporting the potential for malignant transformation of BA into IMA, highlighting KRAS and CDK6 as possible driver genes in this process. These findings contribute to a better understanding of the biological behavior of BA and emphasize the importance of regular imaging follow-up for patients. Furthermore, this case underscores the need for additional studies to improve the understanding of the pathological features, genetic characteristics, and prognosis of BA.